Cell-free homogenates, preparation

Prenyltransferase activities have been studied in C. roseus both at the enzyme level and at the product level. Biosynthetic capabilities were investigated by incubating [1- C]IPP with aliquots of cell-free homogenates prepared from P. aphanidermatum treated and untreated suspension-cultured cells of C. roseus. After elicitation, the total incorporation of IPP into prenyl lipids was decreased, in particular into squalene. But the incorporation of IPP into some (as yet unidentified) compounds was increased (99). The prenyltransferases and subsequent enzyme activities are relatively easily extracted and remain complexed so that the product of one enzyme can be used as a substrate for the next enzyme. With an assay for these enzymes as described in detail in Threlfall and Whitehead (101), about a dozen enzyme activities could be detected in series using cell-free preparations of elicited Tabemaemontana divaricata cells (27). In the elicited C. roseus cells, the activities of IPP isomerase, famesyl diphosphate synthase, squalene synthase, squalene-2,3-epoxidase (and probably also a squalene-2,3-epoxide cyclase) were thus detected. Compared with the control nontreated cells, squalene production seemed to be reduced particularly (99). 

Cell Synchrony and Collection of Mitotic Cells Preparation of Cell-Free Homogenates Nuclear Reassembly... 

Two of the enzymes of the tricarboxylic acid cycle, aconitase and fumarase, catalyze reactions in which water is added reversibly to an unsaturated polycarboxylic acid. Both enzymes exhibit rigid stereospecificity fumarase forms only L-malate from fumarate and forms only fumarate (trans) and not maleate (czs-ethylenedicarboxylic acid), and aconitase reacts with only cis-, not imns-aconitate, and with D-, not L-isocitrate. Citrate is a symmetrical molecule, with no optical isomers, but it will be shown that steric factors also enter into the reaction of this substrate with aconitase. The enzymes of the tricarboxylic acid cycle, in contrast to the glycolytic enzymes, are associated with intracellular granules known as mitochondria. Studies of the individual enzymes have depended to a large extent on the separation of soluble activities from these particles. Aconitase and fumarase are released from the particles very rapidly under mild conditions often in the preparation of cell-free homogenates these activities are largely solubilized, and special care must be taken to demonstrate their origin in mitochondria. 

The work by Scott and Lee 165) on the isolation of a crude enzyme system from a callus tissue culture of C. roseus was followed by studies of Zenk et al. on an enzyme preparation from a cell suspension system which produced indole alkaloids 166). The cell-free preparation was incubated with tryptamine and secologanin (34) in the presence of NADPH to afford ajmalicine (39), 19-epiajmalicine (92), and tetrahydroalstonine (55) in the ratio 1 2 0.5. No geissoschizine (35) was detected. In the absence of NADPH, an intermediate accumulated which could be reduced with a crude homogenate of C. roseus cells in the presence of NADPH to ajmalicine (39). Thus, the reaction for the formation of ajmalicine is critically dependent on the availability of a reduced pyridine nucleotide. 

In 1978, Kutney and co-workers reported on the preparation of cell-free extracts from mature C. roseus plants which produced vindoline (3) (169). Leaves from the plants were homogenized in buffer in the presence of Polyclar AT, and the supernatant produced by centrifugation at 30,000 g was incubated with [2- C]tryptamine and secologanin for 2 hr at 34°C in the presence of FAD and NADPH. Vindoline (3) was isolated by TLC, diluted with cold material, and recrystallized to constant activity. Incorporations were in the range 1.10-1.36%, and no incorporation was observed when boiled enzyme preparation was used. Stemmadenine (36) was not incorporated into vindoline (3) using this preparation. These dramatic results were subsequently published in full (170). 

Figure 5.6 Overview of steps involved in the preparation of a cell-free lysate. The cells are resuspended in a buffered solution at a specified cell density. To this suspension is added a cocktail containing several proteolytic inhibitors. The cells in the suspension are lysed (here by homogenization). Finally the lysate is subjected to a very low speed centrifugation such as 5000g for 10 minutes to remove unbroken cells.

For a detailed investigation of the mechanism of this dealkylation, it became necessary to determine the configuration of the epoxide (92). We developed a cell-free enzyme system prepared from the midguts of the silkworm B. mori. When samples of the supernatant obtained from a homogenate of silkworm guts were incubated separately with the [ H](24i ,28/ )-epoxide (92a) and [ H](24S,28S)-epoxide (92b), only the former was effectively converted to desmosterol (91) [154], However, subsequent in vivo and in vitro studies demonstrated no absolute stereo-... 

The development of present knowledge of the mechanisms of bile acid formation has been possible through a combination of the use of cell-free preparations of liver with the general experimental approach of Bergstrom, Lindstedt, and collaborators. Metabolites formed in the presence of different subcellular fractions of liver homogenates have been isolated and identified. The compounds have been tested as precursors of bile acids in rats with a biliary fistula. The chemical synthesis of a number of unlabeled as well as isotopically labeled steroids has played an important role in these studies. 

Sastry and Kates (1966) measured the incorporation of glycerol [= P]phos-phate into lipid by cell-free preparations from spinach leaves. The microsomal fraction contained most of the activity found in the homogenate, and 97.5% of the incorporated label was found in PA plus LPA. When analyzed separately, LPA accounted for 20-30% of the incorporated label. The... 

Nature of the Suppression Effect of Caraway Seed. Since analytical data demonstrated that the addition of caraway seeds depleted volatile sulfur compounds in sauerkraut, this effect was studied using aqueous extracts of caraway seeds and authentic volatile sulfur compounds in model systems. Cell-free crude caraway seed extracts were prepared by blending the unheated spice seeds with chilled (ice water) potassium phosphate solutions (50 mM, pH 7) containing 0.1 M potassium chloride (1 5, w/w) in a cold room (4°C). Homogenates were then clarified by filtration and centrifugation as described by Chin (21). Data in Figure 7 shows that unheated and heat-treated crude caraway seed extracts (1 mL in a 3 mL buffered solution, pH 8) removed methanethiol (10 pg) from the headsapce in closed serum-type vials (120 mL) at 37 which indicated that both heat-stable and heat-labile entities in caraway seed were involved in the depletion of methanethiol. 

FoMy Acid Oxidation by Cell-Free Preparations. An important development in the study of fatty acid oxidation was made by Munoz and Leloir, who found that homogenates of liver oxidize fatty acids when supplemented with adenine nucleotides, inorganic phosphate, Mg++, cytochrome c (a compoimd involved in electron transport), and a member of the tricarboxylic acid cycle. Others found that washed mitochondria contain all the necessary enzymes for oxidation of fatty acids to either CO2 or acetoacetate, provided the supplements of Munoz and Leloir were added. Such systems are extremely delicate, and for many years it was considered that maintenance of the intact structure of mitochondria was essential for preservation of fatty acid oxidation. 

Hopkins and his collaborators have demonstrated (6, 7) that the presence of GSH in the reduced or oxidized form accelerates the reduction of methylene blue by washed tissue preparations. Oxidation of GSH was found to be catalyzed by liver tissue (8) as well as by cell-free kidney homogenates (9). These findings seemed to bear out the enlightened prediction of... 

The enzymatic synthesis of glutathione was first achieved by Bloch " in cell-free pigeon hver homogenates and in acetone powder preparations of the homogenate. Bloch and his collaborators purified the enzymes involved and present the following mechanism of the synthesis. " ... 

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